Hrp conjugated anti mouse secondary antibody

Cells were lysed in buffer containing 50 mM HEPES (pH 7.6), 75 mM KCl, 1 mM MgCl₂, 1 mM EGTA, 0.1% Triton X‐100, 1 mM DTT, 1 mM PMSF and complete EDTA‐free protease inhibitor cocktail (Roche). Protein concentration was quantified by Bradford assay (Bio‐Rad, USA). For co‐immunoprecipitation, 2 mg protein extract was incubated with 5 μl (~5 μg) rabbit αGFP (Immune Systems) (Immune sera, I) or 2.5 μl (~5 μg) normal rabbit serum (pre‐immune sera, PI) for 30 min and subsequently with 20 μl protein A‐sepharose beads (GE Healthcare, USA) for 45 min. Following centrifugation at 4°C, beads were washed three times in lysis buffer before being heated at 100°C in SDS sample buffer for 6 min.
Precipitates using immune (I) or pre‐immune (PI) sera and whole cell extracts (WCE) were migrated using SDS–PAGE and transferred to nitrocellulose membrane. GFP was detected using anti‐GFP sheep polyclonal (1/5,000, Kevin Hardwick) and HRP‐conjugated anti‐sheep secondary antibody (1/10,000, Jackson ImmunoResearch, USA). Myc was detected using anti‐Myc mouse monoclonal (1/500, Abcam, UK) and HRP‐conjugated anti‐mouse secondary antibody (1/10,000, GE Healthcare, USA). Tubulin was detected by anti‐TAT1 mouse monoclonal (1/1,000, Keith Gull) and HRP‐conjugated anti‐mouse secondary antibody (1/10,000, GE Healthcare, USA). ECL detection was performed using Amersham ECL system (GE Healthcare).

Vero cells and human gastric cancer cells (MKN1, MKN28, and MKN74) were incubated in 10-cm dishes at 2 × 10⁶ cells per dish at 37°C. After 24 hr of incubation, cells were infected with PBS (–) and T-01 (Vero cells: at MOI of 0.01, human gastric cancer cells: at MOI of 0.1) and T-SOCS3 (Vero cells: at MOI of 0.01, human gastric cancer cells: at MOI of 0.1). After 20 hr of incubation at 39.5°C, proteins (30 μg) were applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membrane (Bio-Rad) and blotted for 2 hr with additive monoclonal mouse anti-SOCS3 antibody (diluted 1:1000, R&D systems), anti-pSTAT3 antibody (1:1000, Cell Signaling Technology, Danvers, MA, USA) or with mouse anti-β-actin antibody (diluted 1:2000, Sigma) for an hour. After the membrane rinsed out, a horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (diluted 1:4000, GE healthcare, Piscataway, NJ, USA) was blotted and rinsed. And the, the membrane was exposed to enhanced luminol-based chemiluminescent (ECL) Plus (GE healthcare, Japan).

Reactions contained 100 nM UBA1a, 2.5 μM of UBC2 and UEV1, 100 μM of human wild-type (Boston Biochem) or K63R ubiquitin (2B Scientific) and 5 mM ATP as indicated in 40 μL of ubiquitination assay buffer. Samples were incubated at 37°C for between 0 and 90 min as indicated. For the inhibitor assay, 0–50 μM of NSC697923 (Abcam) was pre-incubated with UBC2 and UEV1 in reaction buffer for 15 min at room temperature prior to addition of UBA1a and ATP. The reaction was then incubated at 37°C for 90 min. The final DMSO concentration in these reactions was 0.5%. Following incubation, reducing sample buffer was added and the samples heated at 90°C for 5 min. Samples were separated by SDS-PAGE and Western blotting carried out using a mouse mono- and polyubiquitinated ubiquitin conjugate (Ubiquigent) or mouse Ub-K63 (ThermoFisher) antibody with HRP-conjugated anti-mouse secondary antibody (GE Healthcare or Promega). In addition to the experimental samples, 100 ng of K63 di-ubiquitin positive control (Ubiquigent) was loaded where indicated.